- Host factor heat-shock protein 90 contributes to baculovirus budded virus morphogenesis via facilitating nuclear actin polymerization. [Journal Article]
- VVirology 2019 Jul 05; 535:200-209
- Hsp90, a highly conserved cellular molecular chaperone, is involved in the life cycle of many viruses. A recent proteomics study revealed that Hsp90 was incorporated into the budded virions (BVs) of …
Hsp90, a highly conserved cellular molecular chaperone, is involved in the life cycle of many viruses. A recent proteomics study revealed that Hsp90 was incorporated into the budded virions (BVs) of baculovirus, we therefore explored the role of Hsp90 during Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection process. The results showed that Hsp90 was essential for AcMNPV BV propagation in cultured cells. Electron microscopy detected that nucleocapsids failed to egress from the nucleus to the cytoplasm for further BV budding. Inactivation of Hsp90 abolished virus-triggered nuclear actin polymerization, a process providing essential driving forces for nucleocapsid egress. Further analyses suggested that this was due to the selectively regulation of the proper protein levels and nuclear accumulation of P40 subunit of host actin related protein 2/3 complex (Arp2/3). Thus, Hsp90 participates in baculovirus BV propagation by facilitating nuclear actin polymerization required for progeny BV production.
- Functional Characterization of the Group I Alphabaculovirus Specific Gene ac73. [Journal Article]
- VSVirol Sin 2019 Jul 17
- Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-, Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group I…
Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-, Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group I and II, and Group I appears to be emerged most recently among all baculoviruses. Interestingly, there are 12 Group I specific genes that are only found in this lineage. Studying these genes is helpful to understand how baculoviruses evolved. Here, we reported the functional analyzing results of ac73, a function unknown Group I specific gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) which is the type species of baculovirus. The AC73 protein encoded by ac73 was found to be expressed during the late stage of infection and incorporated into the nucleocapsids of budded virus (BV) and occlusion-derived virus (ODV). In infected cells, AC73 resided mainly in the ring zone region of the nucleus, and appeared to be assembled into occlusion bodies (OBs). The ac73 knockout and repaired viruses were constructed and studied by in vitro and in vivo infection. Although ac73 was not essential for BV and ODV or OB formation, the BV titer and viral infectivity in insect larvae of ac73 knockout AcMNPV decreased by about 5-8 and 3-4 fold compared to those of wild type virus, respectively, suggesting ac73 contributed to infectious BV production and viral infectivity in vivo. This research provides new insight into the function of this Group I specific gene.
- Functional correlation between subcellular localizations of Japanese encephalitis virus capsid protein and virus production. [Journal Article]
- JVJ Virol 2019 Jul 17
- The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic retic…
The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine-substituted mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine-substituted mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localizations of the flaviviral capsid protein and the formation of infectious viral particles.ImportanceThis study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants, and found that the capsid protein localizes at the nucleolus in a different manner compared to its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of anti-flavivirus small molecules that can target the flavivirus capsid protein.
- The cysteine-rich region of a baculovirus VP91 protein contributes to the morphogenesis of occlusion bodies. [Journal Article]
- VVirology 2019 Jul 03; 535:144-153
- The baculovirus core gene vp91 has been reported to be essential for nucleocapsid assembly and oral infection. Here, we studied the function of vp91 by analyzing its homologue, ha76, in Helicoverpa a…
The baculovirus core gene vp91 has been reported to be essential for nucleocapsid assembly and oral infection. Here, we studied the function of vp91 by analyzing its homologue, ha76, in Helicoverpa armigera nucleopolyhedrovirus (HearNPV). HA76 was expressed at the late stage of HearNPV infection; deletion of ha76 showed that the gene is required for budded virus production. A series of recombinants with truncated ha76 was constructed and analyzed in vitro and in vivo. The results showed that the region encoding the C-terminus of HA76 was essential for nucleocapsid assembly, whereas the N-terminal cysteine-rich region was responsible for oral infection. Electron microscope analyses further showed that the cysteine-rich region contributed to morphogenesis of occlusion bodies (OBs), with amino acids 136-223 of HA76 being critical for this function. The results revealed a novel function of VP91 and suggested that the impact on OB morphogenesis is partially related to oral infectivity.
- The first clawed lobster virus Homarus gammarus nudivirus (HgNV n. sp.) expands the diversity of the Nudiviridae. [Journal Article]
- SRSci Rep 2019 Jul 12; 9(1):10086
- Viral diseases of crustaceans are increasingly recognised as challenges to shellfish farms and fisheries. Here we describe the first naturally-occurring virus reported in any clawed lobster species. …
Viral diseases of crustaceans are increasingly recognised as challenges to shellfish farms and fisheries. Here we describe the first naturally-occurring virus reported in any clawed lobster species. Hypertrophied nuclei with emarginated chromatin, characteristic histopathological lesions of DNA virus infection, were observed within the hepatopancreatic epithelial cells of juvenile European lobsters (Homarus gammarus). Transmission electron microscopy revealed infection with a bacilliform virus containing a rod shaped nucleocapsid enveloped in an elliptical membrane. Assembly of PCR-free shotgun metagenomic sequencing produced a circular genome of 107,063 bp containing 97 open reading frames, the majority of which share sequence similarity with a virus infecting the black tiger shrimp: Penaeus monodon nudivirus (PmNV). Multiple phylogenetic analyses confirm the new virus to be a novel member of the Nudiviridae: Homarus gammarus nudivirus (HgNV). Evidence of occlusion body formation, characteristic of PmNV and its closest relatives, was not observed, questioning the horizontal transmission strategy of HgNV outside of the host. We discuss the potential impacts of HgNV on juvenile lobster growth and mortality and present HgNV-specific primers to serve as a diagnostic tool for monitoring the virus in wild and farmed lobster stocks.
- Identification and Characterization of a Novel Emaravirus Associated With Jujube (Ziziphus jujuba Mill.) Yellow Mottle Disease. [Journal Article]
- FMFront Microbiol 2019; 10:1417
- A previously unreported disease affecting jujube (Ziziphus jujuba Mill.) trees was observed in China (Liaoning province) in 2015 and named jujube yellow mottle disease (JYMD), due to prevalent sympto…
A previously unreported disease affecting jujube (Ziziphus jujuba Mill.) trees was observed in China (Liaoning province) in 2015 and named jujube yellow mottle disease (JYMD), due to prevalent symptoms on the leaves. Diseased plants produced also malformed and discolored fruits. In an attempt to identify the possible causal agent of JYMD, high-throughput sequencing of small RNA libraries was performed and a novel virus, tentatively named jujube yellow mottle-associated virus (JYMaV), was identified and characterized. Six genomic RNA segments of JYMaV were completely sequenced. Each one contains a single open reading frame in the viral complementary strand and two untranslated regions with complementary 5' and 3' terminal ends, thus showing typical features of other negative-stranded RNA viruses. RNA1 (7.1 kb), RNA2 (2.2 kb) and RNA3 (1.2 kb) encode putative proteins that, based on their conserved motifs, have been identified as the RNA dependent RNA polymerase, the glycoprotein and the nucleocapsid protein, respectively. These proteins share significant sequence identity (52.1-70.4%) with proteins encoded by raspberry leaf blotch virus (RLBV). RNA4 (1.5 kb) and RNA5 (1.2 kb) code for two putative 30 K movement proteins also related to the homologous RLBV protein. The functional role of the protein encoded by JYMaV RNA6 remains unknown. These data together with the phylogenetic relationships of JYMaV with other recognized emaraviruses support the proposal that JYMaV is the representative member of a novel species in the genus Emaravirus. In agreement with this proposal, virus-like particles and double-membrane-bound bodies, similar to those previously reported for other emaraviruses, were observed by transmission electron microscopy in extracts and tissues from symptomatic leaves, respectively. A specific RT-PCR-based detection method has been developed and used in a preliminary field survey that provided results strongly supporting the close association of JYMaV with the novel disease.
- Autographa Californica Multiple Nucleopolyhedrovirus P48 (Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles. [Journal Article]
- VSVirol Sin 2019 Jul 10
- Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) gene is essential for the nuclear egress of nucleocapsids and the formation of occlusio…
Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions (ODVs). However, the exact role of p48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In AcMNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore, coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection.
- Self-capping of nucleoprotein filaments protects Newcastle Disease Virus genome. [Journal Article]
- EElife 2019 Jul 10; 8
- Non-segmented negative-strand RNA viruses, such as Measles, Ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids which serve as the template for viral …
Non-segmented negative-strand RNA viruses, such as Measles, Ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode, and the tightly packed structure functions as a seed for nucleocapsid to assemble from both directions and grows into double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations on its loop interface yielded a single-headed unfunctional filament.
- Impact of 5'-end nucleotide modifications of HIV-1 genomic RNA on reverse transcription. [Journal Article]
- BBBiochem Biophys Res Commun 2019 Jul 05
- Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical ro…
Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5'-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (7mG), a hallmark of transcripts generated by RNA polymerase II, at the 5'-end G nucleotide (5'-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3' terminal region of viral RNA (3'-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5'-G with cap or triphosphate form demonstrated that the 5'-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5'-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3'-GGG tract. Lack of apparent contribution of the 3'-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3'-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that 7mG of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3'-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5'-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5'-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5'-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5'-G with 3'-end region of viral RNA.
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- Crystal Structure of the Japanese Encephalitis Virus Capsid Protein. [Journal Article]
- VViruses 2019 Jul 06; 11(7)
- Japanese encephalitis (JE) is inflammation and swelling of the brain caused by the JE virus (JEV), a mosquito-borne member of the Flavivirus family. There are around 68,000 JE cases worldwide each ye…
Japanese encephalitis (JE) is inflammation and swelling of the brain caused by the JE virus (JEV), a mosquito-borne member of the Flavivirus family. There are around 68,000 JE cases worldwide each year, many of which result in permanent brain damage and death. There is no specific treatment for JE. Here we present the crystal structure of the JEV capsid protein, a potential drug target, at 1.98 Å, and compare it to other flavivirus capsid proteins. The JEV capsid has a helical secondary structure (α helixes 1-4) and a similar protein fold to the dengue virus (DENV), the West Nile virus (WNV), and the Zika virus (ZIKV) capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (') through α-helix 1-1', 2-2', and 4-4' interactions. This dimeric form is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix-1 allows the formation of closed and open conformations with possible functional importance. The basic C-terminal pairing of α4-4' forms a coiled-coil-like structure, indicating possible nucleic acid binding functionality. However, a comparison with other nucleic acid interacting domains indicates that homodimerization would preclude binding. This is the first JEV capsid protein to be described and is an addition to the structural biology of the Flavivirus.